Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations

ARTICLE Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations

Dervla M. Connaughton,1,2,46 Rufeng Dai,1,3,46 Danielle J. Owen,4 Jonathan Marquez,5 Nina Mann,1 Adda L. Graham-Paquin,6 Makiko Nakayama,1 Etienne Coyaud,7,8 Estelle M.N. Laurent,7,8 Jonathan R. St-Germain,7 Lot Snijders Blok,9,10,11 Arianna Vino,9 Verena Kla¨mbt,1 Konstantin Deutsch,1 Chen-Han Wilfred Wu,1 Caroline M. Kolvenbach,1 Franziska Kause,1 Isabel Ottlewski,1 Ronen Schneider,1 Thomas M. Kitzler,1 Amar J. Majmundar,1 Florian Buerger,1 Ana C. Onuchic-Whitford,1,12 Mao Youying,1 Amy Kolb,1 Daanya Salmanullah,1 Evan Chen,1 Amelie T. van der Ven,1 Jia Rao,3 Hadas Ityel,1 Steve Seltzsam,1 Johanna M. Rieke,1 Jing Chen,1 Asaf Vivante,1,13 Daw-Yang Hwang,1 Stefan Kohl,1 Gabriel C. Dworschak,1

(Author list continued on next page)

Summary

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CA- KUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

Introduction genes thus far implicated in monogenic forms of CAKUT account for only 14%–20% of cases.3–5 Thus, a significant Congenital anomalies of the kidney and urinary tract (CA- proportion of CAKUT is still molecularly unidentified and KUT) constitute one of the most frequent birth defects, many pathogenic pathways are still elusive.2 To gain causing almost 50% of all cases of end-stage kidney disease further insight into the pathogenesis of human CAKUT, (ESKD) in the first three decades of life.1 In humans, the we performed whole-exome sequencing (WES) in a large identification of 40 monogenic causes of isolated CAKUT international cohort of 551 individuals from different fam- and 153 monogenic causes of syndromic CAKUT has al- ilies with CAKUT. We identified 14 different heterozygous lowed delineation of multiple pathways of human CAKUT mutations in ZMYM2 in 15 unrelated families (19 affected including those involving bone morphogenic protein individuals) with syndromic CAKUT who also had extrare- signaling and retinoic acid signaling.2,3 However, the nal features. ZMYM2 (MIM: 602221), previously known as

1Department of Pediatrics, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; 2Division of Nephrology, Department of Medi- cine, University Hospital - London Health Sciences Centre, Schulich School of Medicine & Dentistry, Western University, 339 Windermere Road, London, ON N6A 5A5, Canada; 3Department of Nephrology, Children’s Hospital of Fudan University, 201102 Shanghai, China; 4Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK; 5Pediatric Genomics Discovery Program, Department of Pediatrics and Genetics, Yale Univer- sity School of Medicine, New Haven, CT 06520, USA; 6Rosalind & Morris Goodman Cancer Research Centre and Department of Biochemistry, McGill Uni- versity, Montreál, QC H3A 1A3, Canada; 7Princess Margaret Cancer Centre, University Health Network & Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; 8Univ. Lille, Inserm, CHU Lille, U1192 - Proteómique Re´ponse Inflammatoire Spectrome´trie de Masse - PRISM, 59000 Lille, France; 9Language and Genetics Department, Max Planck Institute for Psycholinguistics, 6525 XD Nijmegen, the Netherlands; 10Donders Insti- tute for Brain, Cognition and Behaviour, Radboud University, 6500HE Nijmegen, the Netherlands; 11Human Genetics Department, Radboud University Medical Center, 6500HB Nijmegen, the Netherlands; 12Renal Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; 13Tel Aviv University, Faculty of Medicine, Tel Aviv-Yafo 6997801, Israel; 14Amsterdam UMC, University of Amsterdam, Department of Clinical

(Afﬁliations continued on next page) Ó 2020 American Society of Human Genetics.

The American Journal of Human Genetics 107, 727–742, October 1, 2020 727 Tobias Hermle,1 Marie¨lle Alders,14 Tobias Bartolomaeus,15 Stuart B. Bauer,16 Michelle A. Baum,1 Eva H. Brilstra,17 Thomas D. Challman,18 Jacob Zyskind,19 Carrie E. Costin,20 Katrina M. Dipple,21 Floor A. Duijkers,22 Marcia Ferguson,23 David R. Fitzpatrick,24 Roger Fick,25 Ian A. Glass,21 Peter J. Hulick,26 Antonie D. Kline,23 Ilona Krey,15,27 Selvin Kumar,28 Weining Lu,29 Elysa J. Marco,30 Ingrid M. Wentzensen,19 Heather C. Mefford,21 Konrad Platzer,15 Inna S. Povolotskaya,31 Juliann M. Savatt,18 Natalia V. Shcherbakova,31 Prabha Senguttuvan,32 Audrey E. Squire,33 Deborah R. Stein,1 Isabelle Thiffault,34,35,36 Victoria Y. Voinova,31 Michael J.G. Somers,1 Michael A. Ferguson,1 Avram Z. Traum,1 Ghaleb H. Daouk,1 Ankana Daga,1 Nancy M. Rodig,1 Paulien A. Terhal,17 Ellen van Binsbergen,17 Loai A. Eid,37 Velibor Tasic,38 Hila Milo Rasouly,39 Tze Y. Lim,39 Dina F. Ahram,39 Ali G. Gharavi,39 Heiko M. Reutter,40,41 Heidi L. Rehm,42,43 Daniel G. MacArthur,42,43 Monkol Lek,42,43 Kristen M. Laricchia,42,43 Richard P. Lifton,44 Hong Xu,3 Shrikant M. Mane,45 Simone Sanna-Cherchi,39 Andrew D. Sharrocks,4 Brian Raught,7 Simon E. Fisher,9,10 Maxime Bouchard,6 Mustafa K. Khokha,5 Shirlee Shril,1 and Friedhelm Hildebrandt1,*

FIM, ZNF198, or RAMP, is a nuclear zinc finger protein that clinical data, pedigree data, and blood samples from individuals localizes to the nucleus, specifically to the PML body.6 It with CAKUT from worldwide sources using a standardized ques- forms part of a transcriptional complex acting as a core- tionnaire. Informed consent was obtained from the individuals pressor by interacting with different nuclear receptors, and/or the substitute decision maker, as appropriate. The diag- and the LSD1-CoREST-HDAC1 complex on chromatin.7 nosis of CAKUT was made by nephrologists and/or urologists based on relevant imaging. The role of ZMYM2 in kidney and ureter development is Whole-exome sequencing was performed as previously largely unknown and ZMYM2 mutations have not previ- 3 described. Briefly, DNA samples from affected individuals and ously been implicated in kidney disease. unaffected family members were subjected to WES using Agilent SureSelect human exome capture arrays (Life Technologies) with next generation sequencing (NGS) on an Illumina sequencing Material and Methods platform. Sequence reads were mapped against the human reference genome (NCBI build 37/hg19) using CLC Genomics Subjects, Whole-Exome Sequencing, and Variant Workbench (v.6.5.1) software (CLC bio). Mutation analysis was Evaluation performed under recessive, dominant, or de novo models, as pre- Approval for human subjects research was obtained from the Insti- viously published.3,8,9 Mutation analysis was performed by genet- tutional Review Boards of the University of Michigan, Boston icists and cell biologists, who had knowledge regarding clinical Children’s Hospital, and from other relevant local Ethics Review phenotypes, pedigree structure, and genetic mapping, and was Boards. The procedures followed in this study were in accordance in line with proposed guidelines.10,11 Sequence variants remain- with the ethical standards of the responsible committee on hu- ing after WES evaluation were examined for segregation. Filtering man experimentation. Proper informed consent was obtained was performed to retain only alleles with a minor allele frequency from all participants. Following informed consent, we obtained (MAF) < 0.1%, a widely accepted cutoff for autosomal-dominant

Genetics, Meibergdreef 9, 1105 Amsterdam, Netherlands; 15Institute of Human Genetics, University of Leipzig Medical Center, Philipp-Rosenthal- Straße 55, 04103 Leipzig, Germany; 16Department of Urology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; 17Department of Genetics, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, the Netherlands; 18Geisinger, Autism & Developmental Medicine Insti- tute, 100 N Academy Avenue, Danville, PA 17822, USA; 19Department of Clinical Genomics, GeneDx, 207 Perry Pkwy, Gaithersburg, MD 20877, USA; 20Department of Clinical Genetics, Akron Children’s Hospital, One Perkins Square, Akron, OH 44308, USA; 21Division of Genetic Medicine, Department of Pediatrics, University of Washington, 4800 Sand Point Way NE, Seattle, WA 98105, USA; 22Department of Clinical Genetics, University of Amsterdam, 1012 WX Amsterdam, the Netherlands; 23Department of Clinical Genetics, Harvey Institute for Human Genetics, 6701 Charles St, Towson, MD 21204, USA; 24MRC Institute of Genetics & Molecular Medicine, Royal Hospital for Sick Children, The University of Edinburgh, 2XU, Crewe Rd S, Edinburgh EH4 2XU, UK; 25Mary Bridge Childrens Hospital, 316 Martin Luther King JR Way, Tacoma, WA 98405, USA; 26Center for Medical Genetics, NorthShore University HealthSystem, 1000 Central Street, Suite 610, Evanston, IL 60201, USA; 27Swiss Epilepsy Center, Klinik Lengg, Bleulerstrasse 60, 8000 Zu¨rich, Switzerland; 28Department of Pediatric Nephrology, Institute of Child Health and Hospital for Children, Tamil Salai, Egmore, Chennai, Tamil Nadu 600008, India; 29Renal Section, Department of Medicine, Boston University Medical Center, 650 Albany Street, Boston, MA 02118, USA; 30Cortica Healthcare, 4000 Civic Center Drive, Ste 100, San Rafael, CA 94939, USA; 31Veltischev Research and Clinical Institute for Pediatrics of the Pirogov Russian National Research Medical University of the Russian Ministry of Health, Moscow 117997, Russia; 32Department of Pediatric Nephrology, Dr. Mehta’s Multi-Specialty Hospital, No.2, Mc Nichols Rd, Chetpet, Chennai, Tamil Nadu 600031, India; 33Seattle Children’s Hospital, Department of Genetic Medicine, 4800 Sand Point Way NE, Seattle, WA 98105, USA; 34Center for Pediatric Genomic Medicine, Children’s Mercy Hospital, 2401 Gillham Rd, Kansas City, MO 64108, USA; 35Department of Pathology and Laboratory Medicine, Children’s Mercy Hospitals, Kansas City, MO 64108, USA; 36University of Missouri-Kansas City School of Medicine, Kansas City, Missouri, 5000 Holmes St, Kansas City, MO 64110, USA; 37Pediatric Nephrology Department, Dubai Hospital, Dubai, United Arab Emirates; 38Medical Faculty Skopje, University Children’s Hospital, Skopje 1000, North Macedonia; 39Division of Nephrology, Columbia Uni- versity, 630 W 168th St, New York, NY 10032, USA; 40Institute of Human Genetics, University Hospital Bonn, 53127 Bonn, Germany; 41Section of Neona- tology and Pediatric Intensive Care, Clinic for Pediatrics, University Hospital Bonn, Adenauerallee 119, 53313 Bonn, Germany; 42Analytic and Transla- tional Genetics Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA; 43Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA 02142, USA; 44The Rockefeller University, 1230 York Ave, New York, NY 10065, USA; 45Department of Genetics, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06510, USA 46These authors contributed equally to this work *Correspondence: [email protected] https://doi.org/10.1016/j.ajhg.2020.08.013.

728 The American Journal of Human Genetics 107, 727–742, October 1, 2020 disorders.12,13 MAF was estimated using combined datasets incor- Cell Culture and Transfections for cDNA Cloning porating all available data from the 1000 Genomes Project, the Experiments were performed in HEK293 cells purchased from the Exome Variant Server (EVS) project, dbSNP145, the Exome Aggre- American Type Culture Collection (ATCC) biological resource cen- gation Consortium (ExAC), and gnomAD. We filtered to retain ter, unless otherwise stated. For transient transfections, HEK293 variants in genes with a PLI score of >0.3 based on a dominant cells were seeded at 60%–70% confluency in Dulbecco’s modified hypothesis. To predict deleteriousness of variants, we used the Eagle’s medium, supplemented with 10% fetal calf serum and 1% University of Santa Cruz Human Genome Browser for the pres- penicillin/streptomycin and grown overnight. Transfections were ence of paralogous genes, pseudogenes, or misalignments, then carried out using Lipofectamine2000 (Thermo Fisher) and Opti- scrutinized all variants with MAF < 0.1% within the sequence MEM (Thermo Fisher) following the manufacturer’s instructions alignments of the CLC Genomic Workbench software program unless otherwise stated. and employed other web based programs (see Web Resources). Variants were confirmed by Sanger sequencing for segregation Immunofluorescence and Confocal Microscopy in Cell of phenotype with genotype. Lines When trios were available for analysis, data processing of For immunostaining, HEK293 cells were seeded on fibronectin- FASTQs were performed by the Genomics Platform at the Broad coated coverslips in 6-well plates. After 16–24 h, cells were Institute of Harvard and Massachusetts Institute of Technology transiently transfected using Lipofectamine2000 (Thermo Fisher) ac- (Broad Institute). Single-nucleotide polymorphism (SNPs) and in- cording to the manufacturer’s instructions. Experiments were per- sertions/deletions (indels) were jointly called across all samples formed 24–48 h after transfection. Cells were fixed for 15 min using using Genome Analysis Toolkit (GATK) HaplotypeCaller. Default 4% paraformaldehyde and permeabilized for 15 min using 0.5% filters were applied to SNP and indel calls using the GATK Variant Triton X-100. After blocking with 10% donkey serum þ BSA, cells Quality Score Recalibration (VQSR) approach. Lastly, the variants 14 were incubated with primary antibody overnight at 4 C. The were annotated using Variant Effect Predictor (VEP). The following day, cells were incubated in secondary antibody for variant call set was uploaded on to Seqr for analysis of the WES 60 min at room temperature and subsequently stained for 5 min output. with DAPI in PBS. Confocal imaging was performed using the Leica Through collaboration with GeneDx, using genomic DNA from SP5X system with an upright DM6000 microscope, and images were the proband or proband and parent(s), the exonic regions and processed with the Leica AF software suite. Immunofluorescence ex- flanking splice junctions of the genome were captured using periments were repeated at least two times in independent experi- the Clinical Research Exome kit (Agilent Technologies) or the ments. The following antibodies were used for immunostaining: IDT xGen Exome Research Panel v.1.0. Massively parallel (Next- mouse anti-Myc (sc-40, Santa Cruz Biotechnology), rabbit anti- Gen) sequencing was done on an Illumina system with 100 ZMYM2 (ab106624, Abcam), and rabbit anti-ZMYM2 (PA5-28265, base pairs or greater paired-end reads. Reads were aligned to hu- Thermo Fisher), all diluted 1:100. Donkey anti-mouse secondary an- man genome build GRCh37/UCSC hg19 and analyzed for tibodiesconjugated to Alexa Fluor 488 (A-21202, Thermo Fisher) and sequence variants using a custom-developed analysis tool. Addi- donkey anti-rabbit secondary antibody conjugated to Alexa Fluor tional sequencing technology and variant interpretation protocol 15 594(A-21207,Thermo Fisher)were used. Weoriginally hypothesized has been previously described. The general assertion criteria for that missense variants may be present in individuals with a milder variant classification are publicly available on the GeneDx Clin- phenotype. In total, we identified 12 missense variants in ZMYM2 Var submission. (Table S2) in our CAKUT cohort. As such, along with testing loss-of-function variants, we tested the following missense variants in our immunoflurosence (IF) data: p.Val61del (c.181_183del), Control Cohorts p.Glu126Ala (c.377A>C), p.Ile387Ala (c.1159A>G), p.Lys649Arg Variants were also tested for absence from in-house control (c.1946A>G), p.Tyr763His (c.2287T>C), p.Tyr763Leu populations. The control cohort consisted of 100 families with ste- (c.2287_2288delinsTA>CT), p.Gly775Glu (c.2324G>A), p.As- roid-resistant nephrotic syndrome (SRNS) in whom a definitive p997del (c.2990_2992del), and p.Glu1031Lys (c.3091G>A) (Table underlying monogenic cause had already been established. An S2, Figure S1). Please note that although variant p.Tyr763Glnfs*6 additional control cohort of 257 different families with a clinical (c. 2287_2288del) and p.Cys823* (c.2469T>A) were initially diagnosis of nephronophthisis (NPHP) with no genetic cause iden- included in our IF data, these two variants were not included in the tified was also used as previously described.16 analysis of our clinical data as the families in question provided consent to study their variants but did not provide consent to include any clinical data. cDNA Cloning Full-length human ZMYM2 cDNA (cDNA clone HsCD00082148) was subcloned by PCR from full-length cDNA and cDNA clones. Bioluminescence Resonance Energy Transfer (BRET) Expression vectors were generated using LR Clonase (Thermo Assays Fisher) according to the manufacturer’s instructions. The We employed Bioluminescence Resonance Energy Transfer (BRET) following expression vectors were used in this study: pRK5-N- assays to test the interactions between both wild-type and mutant Myc and pcDNA6.2-N-GFP. Mutagenesis was performed using ZMYM2 and wild-type FOXP1. Briefly, wild-type and mutant the QuikChange II XL site-directed mutagenesis kit (Agilent Tech- ZMYM2 constructs were subcloned from the pRK5-N-Myc vector nologies) to generate clones with the ZMYM2 mutations identified into YFP- and rLuc-vectors as previously described.17 Wild-type in each family available at the time of analysis (Table S1). Each FOXP1, FOXP2, and NLS rLuc-constructs were generated in a prior construct was sequenced to verify the correct frame as well as study.17 HEK293T/17 cells were grown in 96-well plates and the proper sequence of any linker introduced during the cloning cultured for 24 h at 37 C with 5% CO2. Cells were transfected us- procedure. ing GeneJuice Transfection Reagent (Merck Millipore) according

The American Journal of Human Genetics 107, 727–742, October 1, 2020 729 to manufacturer’s instructions. At 36 h post-transfection, Endu- via in situ hybridization. For rescue experiments, MOs were in- Ren luciferase substrate (Promega) was added at 60 mM and cells jected in the one-cell embryo with 10 ng in a 2 nanoliter volume. were incubated for 4 h. Emission values were measured using an Subsequently, mRNA corresponding to either wild-type or Infinite 200Pro plate reader (Tecan) using the Blue1 and Green1 fil- mutated ZMYM2 sequences was injected into one cell of the ter sets. Corrected BRET ratios were obtained using the following two-cell embryo with 50 pg in a volume of 2 nanoliters including formula [Green1(experimental condition)/Blue1(experimental condition)] fluorescent tracer to determine the side injected with mRNA for

[Green1(control condition)/Blue1(control condition)]. Further details of subsequent analysis via in situ hybridization. the BRET assay set-up are discussed by Deriziotis et al.17 Whole-Mount In Situ Hybridization Xenopus tropicalis Model We detected Xenopus atp1a1 expression by generating a digoxige- X. tropicalis were housed and cared for in the aquatics facility at nin-labeled antisense probe using the T7 High Yield RNA Synthe- Yale University School of Medicine according to established proto- sis kit (NEB, E2040S) and DIG-dUTP (Sigma) from clone number cols approved by Yale Institutional Animal Care and Use TTpA007m23. Atp1a1 expression serves as a well-established Committee. readout, allowing us to monitor a majority of the pronephric and tubule tissue.23 Embryos were collected at stage 34 and fixed Expression Pattern of zmym2 in MEMFA (1:1:8 103 MEMFA salts, 37% formaldehyde, distilled 3 Previous data have demonstrated RNA expression patterns for water) (10 MEMFA salts: 1 M MOPS, 20 mM EGTA, 10 mM zmym2 in Xenopus (Figure S2).18 In addition, the Papalopulu lab MgSO4) for 1–2 h at room temperature and dehydrated in 100% has deposited images of zmym2 expression in Xenbase (see Web ethanol. Whole-mount in situ hybridization was done as previ- 22 Resources), which are suggestive of expression in the pronephros. ously described. We quantitatively assessed loss of function by We further confirmed expression in a Xenopus model (Figure S2C) measuring the area corresponding to the proximal and intermedi- 24 at stage 34 and although expression is somewhat ubiquitous, we ate tubule in injected and control sides of each embryo. We qual- did find some enrichment in the pronephros and tubule. itatively assessed posterior atp1a1 expression in the pronephric tubule. Rescue efficiency was assessed in the mRNA injected side of embryos in which the contralateral side had morphological abnor- Selection of Variants Tested in Xenopus Modeling malities. Successful rescue was based on the comparison between We prioritized testing certain variants (p.Val61del [c.181_183del], mRNA injected and mRNA un-injected sides of the area of the p.Asp997del [c.2990_2992del], p.Gly257* [c.766_767dupGT], proximal pronephros. Areas were determined by manually delin- p.Gln398* [c.1192C>T], p.Cys536Leufs*13 [c.1607delG], p.Arg eating the bounds of this region in ImageJ. In situ hybridization re- 540* [c.1618C>T], p.Lys812Aspfs*13 [c.2434_2437delAAAG], sults were imaged with a Canon EOS 5d digital camera mounted p.Gly1045Argfs*18 [c.3130_3131dupAA], and p.Gly1045Argfs*33 on a Zeiss discovery V8 stereomicroscope. [c.3130_3131dupAA]) since we hypothesize that these particular variants best represent the varying kinds of disruptions found widely distributed across the ZMYM2 gene locus and so testing these Alcian Blue Staining variants could lead to more informative results about dysfunction. Stage 45 embryos were fixed in 100% ethanol for 48 h at room temperature and then washed briefly in acid alcohol (1.2% HCl in 70% ETOH). A 0.25% alcian blue solution in acid alcohol was used to stain Microinjection of Morpholinos and mRNA in Xenopus the embryos over 48 h at room temperature. Specimens were then Embryos washed in acid alcohol several times, rehydrated into H2O, and We induced ovulation and collected embryos by in vitro fertiliza- bleached for 2 h in 1.2% hydrogen peroxide under a bright light. tion as previously described.19,20 Embryos were raised to stage 34 They were then washed several times in 2% KOH and left rocking or 45 in 1/9MR þ gentamycin. Staging of Xenopus tadpoles was overnight in 10% glycerol in 2% KOH. Samples were processed performed according to Faber and Nieuwkoop.21 Antisense mor- through 20%, 40%, 60%, and 80% glycerol in 2% KOH. Craniofacial pholino oligonucleotides (MO) or mRNAs were injected at either cartilage was then imaged with a Canon EOS 5d digital camera the one-cell stage or into one cell of the two-cell embryo as previ- mounted on a Zeiss discovery V8 stereomicroscope. ously described.22 We employed a splice blocking MO which conceptually leads to exon 3 skipping. The following MOs were used: Control: 50-CCTCTTACCTCAGTTACAATTTATA-30 and Statistical Analysis zmym2 exon-3 intron-3 splice blocking 50-TTGCTGTGGAGGCT- All experiments in the Xenopus model were performed a minimum GAAAAACCT-30 (GeneTools in Web Resources). We performed of three times and numbers stated in graphs are the composite of PCR with primers that span this exon and expect a loss of multiple experiments. Statistical significance of abnormalities and 800 bp. To confirm efficient and specific knockdown of rescues with respect to proximal pronephric size were evaluated ZMYM2, we also tested for rescue of our MO phenotype using using unpaired t tests, while percentages of posterior loss of signal the human mRNA (and individual variants that do not rescue), and abnormal craniofacial cartilage were evaluated using Fisher’s confirming that our knockdown is specific for ZMYM2 in the ex- exact tests using GraphPad Prism v.8. In all figures, statistical < periments presented. We generated in vitro capped mRNA of significance was defined as p 0.05. A single asterisk indicates p < < wild-type and mutated human ZMYM2 from sequences cloned 0.05, while double, triple, and quadruple asterisks indicate p < < into in the pKR5-Myc backboned using the SP6 mMessage ma- 0.005, p 0.0005, and p 0.0001, respectively. Bars in graphs chine kit (Thermo Fisher) following the manufacturer’s instruc- indicate means and standard deviations. tions. MOs for knockdown experiments were injected with 5 ng in a 2 nanoliter volume into one cell of the two-cell embryo. Mice þ This volume included the fluorescent tracer Mini-ruby (Thermo Zmym2 / mice were generated by the Transgenic Core Facility of Fisher) in order to determine the injected side for later analysis the Goodman Cancer Research Centre in a C57BL/6 background

730 The American Journal of Human Genetics 107, 727–742, October 1, 2020 Table 1. Fourteen Heterozygous Mutations in ZMYM2 in 19 Individuals from 15 Families with Syndromic CAKUT Family, Nucleotide Amino Acid Exon Ethnicity, CAKUT Extra-renal Neurologic Individual Change Changea Segregation Gender (Sidednessb) Manifestation Involvement

GM10-21 c.622C>T p.Arg208* 3: 20% mosaic Dutch, F RUS normal skeleton: downslanting hypotonia, ID, palpebral ﬁssures stereotypic movements

GM1-21 c.766_ p.Gly257* 3: de novo USA, M UUT: renal agenesis heart: BAV microcephaly, DD, 767dupGT LUT: hypospadias, skeleton: small hands & hypotonia, tethered cryptorchidism, feet cord chordee, Mu¨llerian duct skin: facial remnants dysmorphsims, convex dysplastic ﬁnger nails, hypoplastic toenails other: feeding problems, oral phase dysphagia, IUGR, growth delay

GM3-21 c.1192C>T p.Gln398* 5: de novo USA, M UUT: RUS normal heart: PDA DD, autistic spectrum LUT: enuresis, skeleton: facial incontinence dysmorphsims other: dental caries

GM9-21 c.1367dupA p.Tyr456* 6: de novo white, F UUT: hypoplastic pelvic skeleton: facial DD, auditory kidney (R) detected by dysmorphisms attention, startle reﬂex, ‘‘reverse phenotyping’’ (triangular face, broad motor stereotypies neck, broad nasal bridge), scoliosis single palmar crease on left, tapered ﬁngers, tapered lower extremities

SSC3-21 c.1607delG p.Cys536 8: pat. NA, Italy, F UUT: UPJO (L) — mild ID Leufs*13 mat. NA

A4730-21 c.1618C>T p.Arg540* 8: de novo Macedonia, UUT: pre-natal skeleton: facial speech delay M hydronephrosis dysmorphism (wide LUT: BL VUR grade 3, interpupillary distance, urethral stricture, mild epicanthal folds, hydrocele testis long nose with a bulbous tip, farsightedness, low set posteriorly rotated ears with a simple helix and protuberant ears), hyper-extensibility of the joints

A1204-21 c.1618C>T p.Arg540* 8: pat. NA, Macedonia, UUT: renal other: hematocolpos, no data mat. NA F agenesis (R) imperforate hymen

GM11-21 c.1623_1627 p.Cys543 8: de novo Moroccan, RUS normal skeleton: DD, mild ID, seizures, delACAGT Valfs*3 M hypertelorsism, small autism, psychosis ears, thick lips, high palate, facial dysmorphsims other: OSA

GM17-21 c.2165T>A p.Leu722* 12: pat. WT, white, M mild hypospadias, skeleton: aplasia cutis DD, autism spectrum mat. Het distal chordee and other: acute disorder (mother has dorsal hooding lymphoblastic ADHD and learning leukemia disability)

GM19-21 c.2338C>T p.Arg780* 13: de novo Switzerland, RUS normal — seizure disorder, MRI M normal, low IQ (85)

GM6-21 c.2434_ p.Lys812Aspfs*18 13:de novo white, F RUS normal heart: VSD, atrial septal microcephaly, DD 2437delAAAG defect, PDA skeleton: short stature 2 SD, short 5th digit with abnormal nails, BL epicanthi, abnormal palmar crease, upturned nasal tip and severe feeding problems

(Continued on next page)

The American Journal of Human Genetics 107, 727–742, October 1, 2020 731 Table 1. Continued Family, Nucleotide Amino Acid Exon Ethnicity, CAKUT Extra-renal Neurologic Individual Change Changea Segregation Gender (Sidednessb) Manifestation Involvement

GM6-22 c.2434_ p.Lys812Aspfs*18 13:de novo white, F RUS normal BL epicanthus, speech delay 2437delAAAG abnormal palmar crease

GM18-12 c.24941G>A IVS15-1G>A Intron 14: pat. white, F RUS NA heart: atrial septal ADHD, autism, WT, mat. het defect behavioral concerns skeleton: epicanthal folds

GM18-22 c.24941G>A IVS15-1G>A white, M brother: RUS NA heart: atrial septal defect

GM18-12 c.24941G>A IVS15-1G>A; white, F mother: heart: atrial septal obligatory splice RUS normal defect site

GM7-21 c.3130_ p.Gly1045 19: pat. WT, white, F RUS normal heart: ECHO normal microcephaly, DD, 3131dupAA Argfs*33 mat. NA skeleton: dysmorphic hypotonia, high facial features, short 5th hyperopia ﬁngers & thumbs, broad big toes, 5th ﬁnger clinodactyly, mild short stature (9th percentile)

GM13-21 c.3176dupA p.Asp1059 20: de novo white, F RUS normal heart: ECHO normal microcephaly, DD, Glufs*2 skeleton: short stature speech delay (3rd percentile), dysmorphic facial features (wide eyebrows, wide interpupillary and intercanthal distance, epicanthal folds, narrow downslanting palpebral ﬁssures, nose with a wide tip, downturned corners of the mouth, small and low set ears with hypoplastic lobule), 5th ﬁnger clinodactyly

GM12-21 c.3246G>A p.Trp1082* 20: pat. NA, white, M RUS: bilateral skeleton: dysmorphic DD, speech delay, mat. het malrotated kidneys and facial features (narrow hypotonia, the right is low lying palpebral ﬁssures, epicanthi and GM12-12 c.3246G>A p.Trp1082* white, F mother RUS NAtelecanthus, small nose intellectually disability and a grooved single philtrum with mild hypoplastic nasal nares), short, thick ﬁngers, [ range of motion joints

Transcript accession number for ZMYM2: GenBank: NM_001190965. Mutations listed in this table were not present in gnomAD database. ADHD, attention deficit hyperactivity disorder; ASD, atrial septal defect; BAV, bicuspid aortic valve; BL, bilateral; DC, disease causing, DD; developmental delay; Del, deleterious; ECHO, echocardiogram; F, female; het, heterozygous; ID, intellectual disability; IUGR, intra-uterine growth retardation; L, left; LUT, lower urinary tract; mat., maternal; M, male; NA, not available; OSA, obstructive sleep apnoea; pat., paternal; PDA, patent ductus arteriosus; PNS, peripheral nervous system; PPH2 score, HumVar Poly- Phen-2 prediction score; R, right; RUS, renal ultrasound; SIFT, sorting tolerant from intolerant; Tol., tolerated; UUT, upper urinary tract; UPJO; ureteropelvic junction obstruction; RUS, renal ultrasound; VACTERL, vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities; VSD, ventricular septal defect; VUR, vesicoureteral reflux; WT, wild type. aUnderline indicates Macedonian founder mutation. bSidedness of CAKUT phenotype given in parentheses.

using a CRISPR-Cas9 targeting approach. For this mouse model, both in humans and mice. A D1 allele was selected and propagated we chose to replicate the truncating mutation in an early exon in C57BL/6 background for three generations prior to phenotyp- found in individual GM1-21 (p. Gly257* [c.766_767dupGT] Table ical analyses. Genotyping was performed by PCR ampliﬁcation us- 1), because it is associated with a strong human phenotype, and ing primers 50-ACCTCCTCCATCTTCTGCAC-30 and 50-AAAA since any resulting mutant protein would be severely truncated, GGTCCAACTCCAGCCT-30 amplicon sequencing. Animals and showing subcellular mislocalization according to cellular assays experiments were kept in accordance with the standards of the an- (Figure S1). To model this frameshift mutation, sgRNA 50-GTTA imal ethics committee of McGill University, and the guidelines of CAACCTTAGAAACAGG-30 was designed against exon 3 found the Canadian Council of Animal Care.

732 The American Journal of Human Genetics 107, 727–742, October 1, 2020 Vesicoureteral Reflux of 1,000 for activation), using higher energy collision induced Vesicoureteral reflux was assessed through methylene blue injec- dissociation (HCD) fragmentation. Dynamic exclusion was acti- tion into the bladder as described.25 Briefly, the urinary tract of vated such that MS/MS of the same m/z (within a range of 10 freshly sacrificed newborns was exposed and the bladder was in- ppm; exclusion list size ¼ 500) detected twice within 5 s were jected using a 30-gauge needle connected to a reservoir filled excluded from analysis for 15 s. For protein identification, Ther- with methylene blue dye (1 mg/mL). To increase hydrostatic pres- mo.RAW files were converted to the .mzXML format using Proteo- sure, the syringe was raised at 5 cm/s to 120 cm. Reflux and ure- wizard,28 then searched using X!Tandem29 and Comet30 against thral voiding pressures were recorded as reservoir height (cm). the human Human RefSeq Version 45 database (containing 36,113 entries). Search parameters specified a parent ion mass Histology tolerance of 10 ppm and an MS/MS fragment ion tolerance of 0.4 Da, with up to 2 missed cleavages allowed for trypsin. Variable Whole urogenital tracts from E18.5 embryos and P0 neonates were modifications of þ16@M and W, þ32@M and W, þ42@N termi- dissected in PBS and fixed overnight in 4% PFA at 4C. Samples were nus, and þ1@N and Q were allowed. Proteins identified with an then processed for paraffin embedding and sectioning (4 mm thick- iProphet cut-off of 0.9 (corresponding to %1% FDR) and at least ness). Tissue sections from each sample were stained with Hematox- two unique peptides were analyzed with SAINT Express ylin & Eosin for tissue analysis. Immunohistofluorescence analyses v.3.6.1.31 Twenty control runs (consisting of BioID conducted on were performed as described.26 The following antibodies were used the same cell type stably expressing the FLAG-BirA* epitope tag for immunostaining: anti-Pax2 (Covance cat# PRB-276P) at 1:200, alone) were collapsed to the four highest spectral counts for anti-Podocalyxin (R&D systems cat# AF1556) at 1:200, anti-Cyto- each prey and compared to the two technical replicates and two keratin 8/18 (Fitzgerald cat# 20R-CP004) at 1:300, and anti- biological replicates of the baits. High confidence interactors Zmym2 (Origene cat# AP08258PU) at 1:100 dilution in PBS. were defined as those with FDR % 0.01. All raw mass spectrometry files have been deposited at the MassIVE archive (see Data and Proximity-Based Biotinylation (BioID) Analysis Code Availability), with accession number ID MSV000085033. Proximity-based biotinylation, or BioID, is a method developed for the characterization of protein-protein interactions in living cells.27 Briefly, Flp-In T-REx 293 cells were stably transfected with pcDNA5 Results FRT/TO Flag-BirA-R118G (FlagBirA*) expression vectors, containing open reading frames for human ZMYM2 (wild type or deletion mu- Mutations of ZMYM2 Cause CAKUT tants) or ZMYM3. Cells at 80% confluence were incubated for 24 h in complete media supplemented with 1 mg/mL tetracycline (Sigma- In pursuit of additional monogenic causes for CAKUT, we 32 Aldrich) and 50 mM biotin (BioShop). Cells were harvested, lysed performed WES in 551 individuals with CAKUT. We de- > (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM tected a heterozygous mutation (p.Arg540* [c.1618C T]) EGTA, 1% Triton X-100, 0.1% SDS, protease inhibitor cocktail, tur- in the gene Zinc Finger MYM-Type Containing 2 (ZMYM2, bonuclease), sonicated twice for 10 s at 35% amplitude (Sonic Dis- GenBank: NM_197968.2) in an affected individual with membrator 500; Fisher Scientific), and centrifuged at 16,000 rpm syndromic CAKUT in an outbred Macedonian family (35,000 3 g) for 30 min at 4 C. Supernatants were passed through (A4730, Table 1). The mutation was de novo and was absent a Micro Bio-Spin Chromatography column (Bio-Rad 732-6204) from control databases ExAC and gnomAD (Table 1, m and incubated with 30 L of high performance streptavidin-Sephar- Figure S3). Direct inspection of sequence alignments ose beads (GE Healthcare) for 3 h at 4C on an end-over-end rotator. from whole-exome data did not yield a mutation in any Beads were pelleted (2,000 rpm, 2 min) and washed six times with of the 40 known isolated human CAKUT genes, the 153 50 mM ammonium bicarbonate (pH 8.3). Washed beads were treated with L-1-Tosylamide-2-phenylethyl chloromethyl ketone known human syndromic CAKUT genes, nor the 185 3 (TPCK)-treated trypsin (Promega) for 16 h at 37C with end-over- known murine CAKUT genes, as previously described. end rotation. After 16 h, another 1 mL of TPCK-trypsin was added The individual, A4730_21, presented with pre-natal hydro- for 2 h and incubated in a water bath at 37C. Supernatants were nephrosis due to a urethral stricture (Table 1). Post-natal ul- lyophilized and stored at 4C. Two biological and two technical rep- trasound revealed bilateral grade three vesicoureteral reflux licates were analyzed using mass spectrometry (below) to identify (VUR) with clinical evidence of a hydrocele. Post WES, ex- high confidence proximity interactors. tra-renal features were noted including facial dysmorphism, hyper-extensibility of the joints, and speech delay Mass Spectrometry Analysis (Figure 1A). Liquid chromatography-electrospray ionization-tandem mass In an unrelated Macedonian family (A1204_21, Table 1), spectrometry (LC-ESI-MS/MS) was conducted as previously we detected the same variant as in the index family 27 described. Briefly, high performance liquid chromatography (p.Arg540* [c.1618C>T]) from our initial cohort of 551 indi- was conducted using a 2 cm pre-column (Acclaim PepMap viduals with CAKUT. This female individual had right renal 50 mm 3 100 mm inner diameter [ID]) and 50 cm analytical col- agenesis. Similar to the index person (A4730_21), additional umn (Acclaim PepMap, 500 mm 3 75 mm diameter; C18; 2 mm; genitourinary tract pathologies were noted: hematocolpos 100 A˚ , Thermo Fisher Scientific), running a 120 min (35,000 3 g) reversed-phase buffer gradient at 225 nL/min on a Proxeon secondary to imperforate hymen (Figure 1A). Due to loss EASY-nLC 1000 pump in-line with a Thermo Q-Exactive HF quad- of follow-up, additional extra-renal or neurological features rupole-Orbitrap mass spectrometer. A parent ion scan was per- could not be assessed. formed using a resolving power of 60,000, then up to the 20 Through collaboration with Columbia University, New most intense peaks were selected for MS/MS (minimum ion count York, we identified family SSC3 with a mutation in

The American Journal of Human Genetics 107, 727–742, October 1, 2020 733 Figure 1. Whole-Exome Sequencing Identifies 14 Heterozygous Loss-of-Function Mutations in ZMYM2 in 15 Families with 19 Affected Individuals (A) Clinical features of individuals with ZMYM2 mutations (see Table 1); family number is shown in the white rectangle. Family GM7: Hypertelorism; simple helix and protuberant ears; 5th fingers and thumbs; 5th finger clinodactyly. Family A4730: Wide eyebrows, mild synophrys, short filtrum; long nose with a bulbous tip; auricle with hypoplastic lobule; hyperexten- sibility of joints. Family GM13: Wide interpupillary distance and intercanthal distance; small auricle; clinodactyly. Family A1204: hematocolpos pre- and post-drainage. Family GM6: (GM6_21) dysmorphic facial features with epicanthi, short 5th digit with hypoplastic nails, abnormal palmar crease and sandal gap toe; (GM6_22) Dysmorphic features – epicanthi. (B) Exon structure of human ZMYM2 cDNA (GenBank: NM_197968.2) and positions of mutations (arrowheads). (C) Protein domain structure of human Zmym2 showing the positions of each of the 14 different heterozygous mutations identified in 15 families (position indicated by the arrows shafts). aa, amino acid; ATG, start codon; NLS, nuclear localization site.

ZMYM2 (Table 1). The proband (SSC3_21) was a female hands and feet with dysplastic/hypoplastic nails, clinodac- with a truncating frameshift mutation (p.Cys536Leufs*13 tyly, and neurological features. Neurological manifesta- [c.1607del]) (Table 1). She had uretero-pelvic junction tions were noted in 14 families (16 affected individuals) obstruction and evidence of intellectual disability (Table 1). and included microcephaly (4/14), developmental delay Using Genematcher,33 we identiﬁed an additional 12 fam- (9/14), intellectual disability (4/14), speech delay (4/14), ilies who carried loss-of-function mutations in ZMYM2 and infantile hypotonia (3/14) (Table 1, Figure 1). (Table 1, Figure 1). All families had extra-renal features of disease or neurological involvement. De Novo Pattern of Inheritance In 8 of 15 families, DNA was available from both parents, Phenotypic Features and for four families a single parental DNA sample was In total, we detected 14 different heterozygous nonsense available. For eight of these families segregation analysis or frameshift mutations of ZMYM2 in 15 families with 19 was consistent with a de novo mutation (Table 1, affected individuals with CAKUT and/or syndromic Figure S3). Germline mosaicism was observed in 1 of the extra-renal features. The phenotypic spectrum included 14 families (GM10). In the two families (GM17 and CAKUT in 7 of 14 families, while all affected individuals GM18) where maternal DNA was available, we were able displayed extra-renal features. Common extra-renal fea- to conﬁrm that the variant was inherited from an affected tures included cardiac defects, facial dysmorphisms, small mother (Table 1). In both cases, the affected mother

734 The American Journal of Human Genetics 107, 727–742, October 1, 2020 Figure 2. Xenopus tropicalis Model of Zmym2 Loss of Function (A) Schematic of the experimental procedure for injection of morpholino into one cell of a two-cell embryo. One side of the embryo is subject to the knockdown, while the other serves as an internal control. (B and C) Representative images and quantitation of decreased pronephric area in one- sided zmym2 morphants. (D and E) Representative images and quantitation of decreased caudal atp1a1 signal in one-sided zmym2 morphants. (F) Schematic of the experimental procedure for injection of morpholino into a one- cell stage embryo. (G and H) Representative images and quantitation of craniofacial dysmorphology in zmym2 morphants, and frequency of rescue of this phenotype in zmym2 morphants co- injected with ZMYM2 mRNA. (I and J) Representative images and quantitation of cloacal exstrophy in zmym2 morphants. (K) Quantitation of proximal pronephric size abnormalities comparing the ratio of proximal pronephric size on the mRNA versus MO only side of an embryo between those injected with mock mRNA, control missense mutants, and those injected with the human ZMYM2 mRNA variants representing truncating mutants. Scale bars depict 500 mm. ****p < 0.0001, **p < 0.005, *p < 0.05 by unpaired t test (C, K) and Fisher’s exact test (E, H, J). Bars indicate mean and standard deviation.

s649Arg (c.1946A>G), p.Tyr763His (c.2287T>C), p.Tyr763Leu (c.2287_ 2288delinsTA>CT), p.Gly775Glu (c.2324G>A), p.Asp997del (c.2990_ displayed neurological manifestations of disease (atten- 2992del), p.Glu1031Lys (c.3091G>A) (Table S2). However, tion deficit hyperactivity disorder [ADHD] and learning the frequency of these variants was similar to the frequency disability), while the affected mother from family GM_18 of missense variants in a control steroid-resistant nephrotic also had evidence of cardiac involvement (Table 1). syndrome and nephronopthisis cohorts (Table S4). In addi- Our findings regarding de novo and mosaic occurrence tion, these variants all retained their nuclear localization strongly suggest that heterozygous truncating mutations and transcriptional repression properties, making causality of ZMYM2 convey infertility or interfere with germline unlikely (Figures S1 and S4). transmission. Allelic frequency data in gnomAD further support this hypothesis. All mutations observed in the Zmym2 Knockdown in X. tropicalis Leads to Defects in affected individuals of this study are absent from control Renal and Craniofacial Development populations (ExAC and gnomAD) while other ZMYM2 To evaluate the deleteriousness of the mutations observed loss-of-function variants are extremely rare, with only 31 in vivo, we generated an X. tropicalis model of zmym2 loss- such variants recorded, 27 of which only occurred once of-function (Figures 2 and S5). At the two-cell stage, Xeno- in approximately 270,000 indiviudals (Table S3). pus embryos were injected with a morpholino oligo (MO) We originally hypothesized that missense variants may targeting zmym2. While one side of the embryo developed be present in persons with a milder phenotype, as has previ- from the un-injected cell and served as an internal control, ously been described for other genes implicated in CAKUT.34 the contralateral side developed from the MO injected cell In total, we identified 12 missense variants in ZMYM2 (Table (Figures 2A–2E). At stage 34, in situ hybridization for the S2) in our CAKUT cohort. As such, we tested the following pronephric marker atp1a1 was employed to assess for de- missense variants in our IF data: p.Val61del (c.181_183del), fects in pronephros morphology in response to MO-medi- p.Glu126Ala (c.377A>C), p.Ile387Val (c.1159A>G), p.Ly- ated knockdown. Specifically, the posterior segment of the

The American Journal of Human Genetics 107, 727–742, October 1, 2020 735 pronephric tubules were evaluated for the level of atp1a1 CAKUT-like defects including hydroureter as well as duplex expression, which characteristically meets the proctodeum and cystic kidneys at E18.5 (Figures 3A–3C). When tested of the embryo at its caudal aspect. Additionally, the prox- for vesicoureteral reflux, 25% of newborns had a reflux imal region of the embryonic pronephri corresponding phenotype, including a majority at or below voiding pres- to the proximal and intermediate tubule in humans24 sure (Figures 3D and S6). None of those malformations were quantified to determine zmym2 knockdown effects were observed in wild-type animals. The presence of CA- þ on these structures. Compared to control sides of the em- KUT-like phenotypes in Zmym2 / animals is compatible bryos, sides with MO knockdown of zmym2 demonstrated with the low but widespread expression levels of Zmym2 loss of caudal atp1a1 signal and decreased proximal pro- in the developing kidney (Figure S7). Immunofluorescence nephric area, suggesting that zmym2 has a specialized analysis of E18.5 kidneys without overt malformations þ role in pronephric development in a subset of regions. showed normal tissue architecture in Zmym2 / animals Quantification of these experiments revealed a loss of pos- (Figure 3E). No additional phenotypes were observed in þ terior atp1a1 signal in 30% of embryos on the side repre- Zmym2 / animals. senting zmym2 knockdown (Figure 2C). To assess the functionality of ZMYM2 mutations discov- ered in individuals, we employed unilateral injection of Intracellular Localization of Truncated ZMYM2 Proteins zmym2 morphants with wild-type or variant ZMYM2 ZMYM2 is a MYM type zinc finger protein that harbors two mRNA reflecting individual sequences (p.Gly257* putative nuclear localization signals (NLS) and ten MYM [c.766_767dupGT], p.Gln398* [c.1192C>T], p.Cy- type zinc fingers (Figures 1B–1G).35 The loss-of-function s536Leufs*13[c.1607delG], p.Arg540* [c.1618C>T], p.Ly- variants that we identified were frameshifts and/or stop- s812Aspfs*18 [c.2434_2437delAAAG], and p.Gly1045 gains prior to the final exon, and therefore mutant Argfs*33 [c.3130_3131dupAA], in addition to two missense mRNA transcripts are predicted to undergo nonsense- variants, p.Val61del [c.181_183del] and p.Asp997del mediated decay (NMD), suggesting a haploinsufficiency [c.2990_2992del]). This approach revealed that only wild- as the most likely mechanism for the associated disorder. type mRNA resulted in rescue of area size (Figures 2F–2K Nonetheless, we used cell-based assays to assess the func- and S5). In contrast, unilateral injection of mRNA reflect- tional properties of truncated protein that might result ing the truncating variants, that we identified in individ- from escaping this NMD process, comparing them to uals, resulted in little to no restoration of proximal pro- wild-type ZMYM2 protein, as well as missense variants. nephric area (Figure 2K). Transfection of expression constructs in HEK293 cells re- We then allowed zmym2 knockdown tadpoles to develop vealed that, whereas the wild-type and missense ZMYM2 further in order to identify phenotypes that may not be protein (p.Val61del [c.181_183del], p.Glu126Ala visible early on. At these later stages, protrusion of tissue [c.377A>C], p.Ile387Val [c.1159A>G], p.Lys649Arg through the primitive cloaca was also apparent in 33% of [c.1946A>G], p.Tyr763His [c.2287T>C], p.Tyr763Leu zmym2 MO-injected embryos and none of the control [c.2287_2288delinsTA>CT], p.Gly775Glu [c.2324G>A], MO-injected embryos. In addition, craniofacial abnormal- p.Asp997del [c.2990_2992del], and p.Glu1031Lys ities became readily apparent. To assess these abnormal- [c.3091G>A]) was translocated to the nucleus, the ities further, we used Alcian blue staining to delineate the ZMYM2 truncated proteins (p.Gly257* [c.766_767dupGT], cartilage morphology within stage 45 embryos. In 73% of p.Gln398* [c.1192C>T], p.Cys536Leufs*13 [c.1607delG], zmym2 MO-injected embryos, gross morphological anom- p.Arg540* [c.1618C>T], p.Lys812Aspfs*18 [c.2434_ alies were observed as compared to 3% of control MO-in- 2437delAAAG], p.Gly1045Argfs*33 [c.3130_3131dupAA]) jected embryos. The frequency of this phenotype in MO- remained primarily localized to the cytoplasm (Figures injected embryos was reduced to 27% via reintroduction 4A and S1). of wild-type ZMYM2 mRNA (Figure 2K). Noteworthy is For three of the truncations (p.Gly257* that variant p.Lys812Aspfs*18 actually worsens the pheno- [c.766_767dupGT], p.Gln398* [c.1192C>T], p.Arg540* type when used in a rescue experiment, which is suggestive [c.1618C>T]), the proteins showed exclusively cyto- that this variant has the potential to function as dominant plasmic patterns in all cells tested. However, for the other negative.These findings are consistent with a pathologic four truncations that we tested (p.Try763Glnfs*6 role for heterozygous ZMYM2 truncating mutations in [c.2287_2288del], p.Cys812Aspfs*18 [c.2434_2437de- urinary tract abnormalities and facial dysmorphisms lAAAG], p.Cys823* [c.2469T>A], and p.Gly1045Argfs*33 observed in individuals with this disorder. [c.3130_3131dupAA]), protein localization was partially nuclear in a subset of cells, despite the loss of one or Mouse Model both known NLSs (p.1038–1049 and p.1250–1284) in the To further validate the causal role of ZMYM2 in CAKUT- truncated proteins. We hypothesize that an additional related developmental defects, we generated a mouse functional NLS lies between p.540 and p.763, thereby ac- model recapitulating the frameshift mutation found in counting for the partial nuclear staining for these trun- exon 3 of individual GM1-21 using CRISPR-Cas9 gene tar- cated proteins (see Supplemental Discussion and þ geting (Figure S6). Zmym2 / mice showed a spectrum of Figure S8 for further details).

736 The American Journal of Human Genetics 107, 727–742, October 1, 2020 Figure 3. Array of CAKUT Phenotypes þ/ Observed in a Zmym2 Mutant Mouse Model þ Zmym2 / mice heterozygous for a frameshift mutation in exon 3 were analyzed at embryonic stage E18.5 and post-natal stage P0. (A) Percentages of mice with given CAKUT þ phenotype observed in Zmym2 / pups and their wild-type littermates. Statistical analysis was done using a binomial test. For CAKUT, hydroureter, duplex, and cystic kidneys phenotypes were compiled from þ þ þ Zmym2 / (n ¼ 35) and Zmym2 / (n ¼ 37). Vesicoureteral reflux (VUR) was as- þ þ sessed from Zmym2 / (n ¼ 25) and þ Zmym2 / (n ¼ 20). Note, some animals harbored more than one CAKUT phenotype. (B) Dissected E18.5 and P0 urogenital sys- þ þ þ tem of Zmym2 / and Zmym2 / mice demonstrating gross CAKUT phenotypes including hydroureter, hydronephrosis, duplex systems, and cystic kidneys, respectively. (C) Haemotoxylin and Eosin staining of tis- þ þ sue sections derived from Zmym2 / and þ Zmym2 / urogenital systems. (D) Intravesical dye injection showing þ þ vesicoureteral reflux in Zmym2 / and þ Zmym2 / P0 mice. (E) Immunohistofluorescence analysis of E18.5 kidneys reveals no overt difference in cap mesenchyme and ureter tips (Pax2) nor in podocytes (podocalyxin) between þ þ þ Zmym2 / and Zmym2 / kidneys.

donor and acceptor proteins come into close proximity of each other in co- transfected cells, energy transfer takes place from RLuc to YFP, which can be quantified by monitoring emission. Consistent with earlier studies, we were able to thereby demonstrate interac- ZMYM2 Interaction with FOXP1 tions between wild-type ZMYM2 and wild-type FOXP1, In prior work, Bekheirnia et al. identified de novo mutations FOXP2, or ZMYM2 (homodimerization). All three truncated in FOXP1 (MIM: 613670) in six families with syndromic CA- versions of ZMYM2 that we tested with BRET (p.Gly257* KUT,36 while Estruch et al. demonstrated that ZMYM2 is able [c.766_767dupGT], p.Gln398* [c.1192C>T], p.Arg540* to interact with different FOXP transcription factors.37 As [c.1618C>T]). showed impaired interaction with FOXP1 noted above, transcripts with truncating ZMYM2 variants and FOXP2 when compared with wild-type ZMYM2 found in affected individuals of this study most likely un- (Figure S9). dergo NMD. However, if such transcripts (partially) escape NMD, there remains the question of whether truncated Expanding the ZMYM2 Interactome ZMYM2 proteins would retain the ability to interact with We employed proximity-dependent biotin identification FOXP transcription factors. To test this possibility, we used (BioID: 22412018) to characterize the ZMYM2 protein Bioluminescence Resonance Energy Transfer (BRET), a live- interaction landscape. In total, 123 high-confidence cell assay system for detecting putative protein-protein inter- (FDR % 1%) ZMYM2 proximity interactors were identified actions.17 In these experiments, either wild-type FOXP1 or (Table S5; all raw data available at MassIVE archive). The FOXP2 (MIM: 602081) was expressed as a renilla luciferase interactome is significantly enriched in DNA binding (RLuc) fusion protein, to function as a donor in the assay. transcription factors (p ¼ 8.3 3 10 22), transcriptional Different variant ZMYM2 constructs were expressed as co-repressors (p ¼ 3.5 3 10 7), and proteins linked to fusion proteins with YFP, to function as acceptors. If the chromatin regulation (p ¼ 9.26 3 10 14), chromatin

The American Journal of Human Genetics 107, 727–742, October 1, 2020 737 Figure 4. Functional Characterization of ZMYM2 Variants and Identiﬁcation of Protein-Protein Interaction Partners of ZMYM2 as Candidates for Monogenic Causes of CAKUT (A) Representative immunoﬂuoroscence images following overexpression of myc labeled cDNA constructs for mock, wild-type ZMYM2 (hsMYC_wtZMYM2), and cDNA representing mutation p.Arg540* (detected in A4730 and A1204) showing mislocalization of truncated protein to the cytoplasm rather than the nucleus. (B) BioID of human wild-type ZMYM2 expressed in Flp-In T-REx 293 cells yields 123 proximity interaction partners. Interactors are grouped according to protein complex, intracellular localization, shared protein domain, or function. Edge size is proportional to total peptide counts. (C) All 73 candidate genes resulting from the BioID experiments were evaluated for heterozygous mutations in 551 families with CAKUT using the American College of Medical Genetics criteria for deleteriousness. (D) ZMYM3 variants in families B1287_21 and B2323_21 as a potential candidate gene in CAKUT pathogenesis. CAKUT, congenital anomalies of the kidney and urinary tract; c. change, nucleotide change; Cov., coverage; gnomAD, genome aggregation; Miss., missense; Mut. Taster, Mutation Taster; NS, nephrotic syndrome; p. change, amino acid change, Poly2, Polymorphism Phe- notyping v2; SIFT, Sorting Intolerant From Tolerant; WES, whole-exome sequencing; Zyg, zygosity; Mm, Mus musculus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Pk, Paramormyrops kingsleyae; Ci, Ciona intestinalis; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae.

organization (p ¼ 5.91 3 10 14), and the SUMO system (p EHMT1, EHMT2, TFDP1), components of the DREAM ¼ 6.7 3 10 05). A number of previously reported ZMYM2 (MYBL2, LIN52, LIN54, LIN9), ChAHP (CHD4, CBX1, interactors were identified in our analysis:7 the ADNP), and TFIIIC (GTF3C1-4) complexes, and a large LSD1(KDM1A)-CoREST (Corum complexes 633 and number of zinc finger-containing DNA binding proteins 1492),38 HDAC139 and HDAC2 and many of their known as high-confidence ZMYM2 interactors (Table S5). Consis- interacting partners (e.g., Corum 632: HDAC1, HDAC2, tent with a previous report,6 ZMYM2 BioID also identified KDM1A/LSD1, GTF2I, GSE1/KIAA0182, PHF21A/BHC80, a number of PML body components (PML, BLM, DAXX, RCOR1, RCOR2, RCOR3, ZNF217, ZMYM2, and HIPK2, MORC3, RAD50, ZNF24). Finally, consistent with ZMYM3), and the transcription factors FOXP137 and recent reports identifying ZMYM2 as a SUMO binding SIX4 (Table S5). Consistent with a recent report linking protein,41,42 we also detected high-confidence interac- ZMYM2 function to endogenous retrovirus silencing,40 tions with a number of SUMO conjugation system com- BioID also identified components of the epigenetic ponents (PIAS1, PIAS2, PIAS4, ZNF451, SIMC1/C5orf25) repressor complex SETDB1-ATF7IP (SETDB1, ATF7IP, (Table S5). Together, these data link ZMYM2 to transcrip- C11orf46), the non-canonical polycomb complex E2F6/ tional repression and the epigenetic regulation of hetero- PRC1.6 (PCGF6, E2F6, MGA, L3MBTL2, L3MBTL3, chromatin and generate excellent candidates for

738 The American Journal of Human Genetics 107, 727–742, October 1, 2020 additional genes potentially involved in monogenic forms Furthermore, by generating a mouse model of heterozy- of CAKUT (Figure S10). gous Zmym2 disruption, we recapitulated the human CA- Our ZMYM2 BioID identified two transcription factor KUT phenotype, confirming the importance of ZMYM2 proteins that have been linked to kidney development, in renal development and as a cause of CAKUT in humans SIX443–45 and FOXP1.36 SIX4 was shown to work together when mutated. Finally, we generate independent evidence with the related SIX1 protein to regulate gene expression confirming that mutant ZMYM2 leads to loss of interac- in metanephric mesenchyme,44 while SIX1 mutations tion with FOXP1, a transcription factor already linked to cause CAKUT in humans.46 To characterize how the CAKUT, as well as uncovering a potential interaction ZMYM2 protein interaction landscape is affected by CAKUT with ZMYM3, which we suggest may represent a method truncations, p.Gly257* (c.766_767dupGT), p.Gln398* to identify candidate genes involved in this disorder. (c.1192C>T), p.Cys536Leufs*13 (c.1607delG), and p.Ly- ZMYM2, also known as FIM, ZNF198, or RAMP, is a nu- s812Aspfs*18 (c.2434_2437delAAAG) ZMYM2 variant pro- clear zinc finger protein that contains 1,377 amino acids teins were also subjected to BioID (Table S5 and with a molecular mass of 150 kDa.47,48 ZMYM2 localizes Figure S11). These mutants lost the vast bulk of interactions to the nucleus, specifically the PML body,6 where it has detected with the wild-type protein, including the interac- been characterized as a corepressor of transcription by in- tion with FOXP1, consistent with the data from BRET teracting with different nuclear receptors, and the LSD1- assays. CoREST-HDAC1 complex on chromatin.7 A recent report has also linked ZMYM2 to silencing of endogenous retro- 40 ZMYM3 Variants as Potential Candidates in CAKUT virus sequences. However, the importance of ZMYM2 Pathogenesis for kidney and ureter development was largely unknown We hypothesized that, like FOXP1, the genes encoding and despite a role in myeloproliferative disorder as a fusion other ZMYM2 interacting partners could also represent protein, ZMYM2 mutations have not previously been candidate genes for involvement in CAKUT. Indeed, exam- implicated in human disease. ination of WES data from our cohort of 551 individuals with ZMYM2 has previously been shown to interact with 37 CAKUT (Figure 4C, Table S6), revealed two male individuals FOXP transcription factors, and mutations in FOXP1 with hemizygous variants in ZMYM3 (MIM: 300061, were recently identified in individuals with syndromic CA- 36 p.Gly673Asp [c.2018G>A] and p.Val866Met [c.2596G>A], KUT. Given that they are all located prior to the final Figures 4D and 4E). Consistent with our ZMYM2 data, BioID exon of the gene, the truncating and stop gain ZMYM2 var- of the ZMYM3 protein yielded a reciprocal interaction with iants found in our study will most likely undergo NMD ZMYM2, and an interactome largely overlapping with that in vivo, preventing their translation and leading to of ZMYM2 (68% overlap; Table S5), including components ZMYM2 haploinsufficiency. However, as demonstrated in of the LSD1-CoREST, ChAHP, DREAM, and TFIIIC com- our Xenopus model, some variants resulted in worsening plexes. While further investigation of the functional role the phenotype, which raises the possibility that some var- of these variants in ZMYM2 pathogenesis is necessary, these iants may in fact function as dominant negative rather methods reveals strategies to identifiy variants in poten- than haploinsufficiency. Furthermore, our BRET assays tially novel target genes involved in the mechanism of dis- indicate that if aberrant transcripts escape NMD, there ease development in CAKUT. would be expression of truncated versions of ZMYM2 that are unable to interact with either FOXP1 or FOXP2. These findings are also supported by our BioID data, again Discussion indicating a loss of FOXP1 interaction for truncated ZMYM2 proteins. In summary, here we describe the discovery of predomi- In addition, the BioID data identified multiple ZMYM2 nantly de novo loss-of-function mutations of ZMYM2 as interactors, including members of the LSD1-COREST- an autosomal-dominant cause of human syndromic HDAC1 pathway, suggesting that the broader ZMYM2 CAKUT. Consistent with its known nuclear function,6 we interactome, which include DNA binding transcription demonstrate that wild-type ZMYM2 is located in the nu- factors, transcriptional co-repressors, and proteins linked cleus, whereas truncated ZMYM2 proteins can mislocalize to chromatin regulation and organization may represent to the cytoplasm. By expression and morpholino knock- potential candidate genes in urinary tract malforma- down experiments in Xenopus larvae, we confirm that tion.7 Given the observation that either FOXP1 or ZMYM2 plays critical roles in kidney and craniofacial ZMYM2 loss-of-function mutations can cause CAKUT, development. In addition, the renal phenotype of Xenopus the other genes in this interactome could also be consid- morphants was rescued by wild-type but not mutant ered as candidates for involvement in the disorder. mRNA, consistent with pathogenicity for the alleles that Further work is now required to determine the role of we identified in individuals. Interestingly, one of the vari- these potential interactors in the pathogenesis of kidney ants resulted in worsening the phenotype, suggesting the malformation in individuals with ZMYM2 mutations. potential for it to yield dominant-negative effects, if the Mechanistically, further studies will help elucidate how variant transcript escapes NMD in individual cells. ZMYM2 mutations lead to the development of CAKUT,

The American Journal of Human Genetics 107, 727–742, October 1, 2020 739 giving novel insights into the biological basis of the 3. van der Ven, A.T., Connaughton, D.M., Ityel, H., Mann, N., disorder. Nakayama, M., Chen, J., Vivante, A., Hwang, D.Y., Schulz, J., Braun, D.A., et al. (2018). Whole-Exome Sequencing Identifies Causative Mutations in Families with Congenital Anomalies Data and Code Availability of the Kidney and Urinary Tract. J. Am. Soc. Nephrol. 29, 2348–2361. All raw mass spectrometry files have been deposited at the 4. Weber, S., Moriniere, V., Knu¨ppel, T., Charbit, M., Dusek, J., MassIVE archive with accession number ID Ghiggeri, G.M., Jankauskiene´, A., Mir, S., Montini, G., Peco- Antic, A., et al. (2006). Prevalence of mutations in renal devel- MSV000085033. opmental genes in children with renal hypodysplasia: results Human ZMYM2 full-length protein is GenBank: of the ESCAPE study. J. Am. Soc. Nephrol. 17, 2864–2870. NM_197968.2. 5. Rasmussen, M., Sunde, L., Nielsen, M.L., Ramsing, M., Pe- Xenopus Zmym2 full-length protein is GenBank: tersen, A., Hjortshøj, T.D., Olsen, T.E., Tabor, A., Hertz, J.M., NM_001123434.1. Johnsen, I., et al. (2018). Targeted gene sequencing and whole-exome sequencing in autopsied fetuses with prenatally diagnosed kidney anomalies. Clin. Genet. 93, 860–869. Supplemental Data 6. Kunapuli, P., Kasyapa, C.S., Chin, S.-F., Caldas, C., and Cowell, Supplemental Data can be found online at https://doi.org/10. J.K. (2006). ZNF198, a zinc finger protein rearranged in myelo- 1016/j.ajhg.2020.08.013. proliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML. Exp. Cell Res. 312, 3739– Revised: April 2, 2020 3751. Accepted: August 14, 2020 7. Gocke, C.B., and Yu, H. (2008). ZNF198 stabilizes the LSD1- Published: September 4, 2020 CoREST-HDAC1 complex on chromatin through its MYM- type zinc fingers. PLoS ONE 3, e3255. 8. Warejko, J.K., Tan, W., Daga, A., Schapiro, D., Lawson, J.A., Web Resources Shril, S., Lovric, S., Ashraf, S., Rao, J., Hermle, T., et al. (2018). Whole Exome Sequencing of Patients with Steroid- 1000 Genomes Browser, https://www.internationalgenome.org/ Resistant Nephrotic Syndrome. Clin. J. Am. Soc. Nephrol. 1000-genomes-browsers 13, 53–62. Conifer software, http://conifer.sourceforge.net 9. Vivante, A., Hwang, D.Y., Kohl, S., Chen, J., Shril, S., Schulz, J., Database of genomic variants, http://projects.tcag.ca/variation van der Ven, A., Daouk, G., Soliman, N.A., Kumar, A.S., et al. Ensembl Genome Browser, http://www.ensembl.org (2017). Exome Sequencing Discerns Syndromes in Patients Exome Variant Server, https://evs.gs.washington.edu/EVS from Consanguineous Families with Congenital Anomalies Exome Aggregation Consortium, http://exac.broadinstitute.org of the Kidneys and Urinary Tract. J. Am. Soc. Nephrol. 28, GenBank, https://www.ncbi.nlm.nih.gov/genbank/ 69–75. GeneDx ClinVar submission, https://www.ncbi.nlm.nih.gov/ 10. MacArthur, D.G., Manolio, T.A., Dimmock, D.P., Rehm, H.L., clinvar/submitters/26957/ Shendure, J., Abecasis, G.R., Adams, D.R., Altman, R.B., Anto- Genematcher, https://genematcher.org/ narakis, S.E., Ashley, E.A., et al. (2014). Guidelines for investi- GeneTools, https://www.gene-tools.com/ gating causality of sequence variants in human disease. Na- gnomAD browser beta, https://gnomad.broadinstitute.org/ ture 508, 469–476. HGMD, https://portal.biobase-international.com 11. Richards, C.S., Bale, S., Bellissimo, D.B., Das, S., Grody, W.W., MassIVE archive, https://massive.ucsd.edu/ProteoSAFe/static/ Hegde, M.R., Lyon, E., Ward, B.E.; and Molecular Subcommit- massive.jsp tee of the ACMG Laboratory Quality Assurance Committee MutationTaster, http://www.mutationtaster.org (2008). ACMG recommendations for standards for interpreta- OMIM, https://www.omim.org/ tion and reporting of sequence variations: Revisions 2007. PolyPhen2, http://genetics.bwh.harvard.edu/pph2 Genet. Med. 10, 294–300. Renal Genes, http://www.renalgenes.org 12. Bamshad, M.J., Ng, S.B., Bigham, A.W., Tabor, H.K., Emond, Seqr, https://seqr.broadinstitute.org/ M.J., Nickerson, D.A., and Shendure, J. (2011). Exome Sorting Intolerant From Tolerant (SIFT), http://sift.jcvi.org sequencing as a tool for Mendelian disease gene discovery. UCSC Genome Browser, https://genome.ucsc.edu Nat. Rev. Genet. 12, 745–755. Xenbase, http://www.xenbase.org 13. Lee, H., Deignan, J.L., Dorrani, N., Strom, S.P., Kantarci, S., Quintero-Rivera, F., Das, K., Toy, T., Harry, B., Yourshaw, M., et al. (2014). Clinical exome sequencing for genetic identifica- References tion of rare Mendelian disorders. JAMA 312, 1880–1887. 1. Vivante, A., Kohl, S., Hwang, D.Y., Dworschak, G.C., and Hil- 14. Lek, M., Karczewski, K.J., Minikel, E.V., Samocha, K.E., Banks, debrandt, F. (2014). Single-gene causes of congenital anoma- E., Fennell, T., O’Donnell-Luria, A.H., Ware, J.S., Hill, A.J., lies of the kidney and urinary tract (CAKUT) in humans. Pe- Cummings, B.B., et al.; Exome Aggregation Consortium diatr. Nephrol. 29, 695–704. (2016). Analysis of protein-coding genetic variation in 2. van der Ven, A.T., Vivante, A., and Hildebrandt, F. (2018). 60,706 humans. Nature 536, 285–291. Novel Insights into the Pathogenesis of Monogenic Congen- 15. Retterer, K., Juusola, J., Cho, M.T., Vitazka, P., Millan, F., Gibel- ital Anomalies of the Kidney and Urinary Tract. J. Am. Soc. lini, F., Vertino-Bell, A., Smaoui, N., Neidich, J., Monaghan, Nephrol. 29, 36–50. K.G., et al. (2016). Clinical application of whole-exome

740 The American Journal of Human Genetics 107, 727–742, October 1, 2020 sequencing across clinical indications. Genet. Med. 18, 696– 32. Otto, E.A., Hurd, T.W., Airik, R., Chaki, M., Zhou, W., Stoetzel, 704. C., Patil, S.B., Levy, S., Ghosh, A.K., Murga-Zamalloa, C.A., 16. Kitzler, T.M., Schneider, R., Kohl, S., Kolvenbach, C.M., Con- et al. (2010). Candidate exome capture identifies mutation naughton, D.M., Dai, R., Mann, N., Nakayama, M., Majmun- of SDCCAG8 as the cause of a retinal-renal ciliopathy. Nat. dar, A.J., Wu, C.W., et al. (2019). COL4A1 mutations as a po- Genet. 42, 840–850. tential novel cause of autosomal dominant CAKUT in 33. Sobreira, N., Schiettecatte, F., Valle, D., and Hamosh, A. humans. Hum. Genet. 138, 1105–1115. (2015). GeneMatcher: a matching tool for connecting investi- 17. Deriziotis, P., Graham, S.A., Estruch, S.B., and Fisher, S.E. gators with an interest in the same gene. Hum. Mutat. 36, (2014). Investigating protein-protein interactions in live cells 928–930. using bioluminescence resonance energy transfer. J. Vis. Exp. 34. Kohl, S., Hwang, D.Y., Dworschak, G.C., Hilger, A.C., Saisawat, https://doi.org/10.3791/51438. P., Vivante, A., Stajic, N., Bogdanovic, R., Reutter, H.M., Ke- 18. Neilson, K.M., Pignoni, F., Yan, B., and Moody, S.A. (2010). hinde, E.O., et al. (2014). Mild recessive mutations in six Developmental expression patterns of candidate cofactors Fraser syndrome-related genes cause isolated congenital for vertebrate six family transcription factors. Dev. Dyn. 239, anomalies of the kidney and urinary tract. J. Am. Soc. Neph- 3446–3466. rol. 25, 1917–1922. 19. del Viso, F., Bhattacharya, D., Kong, Y., Gilchrist, M.J., and 35. Ollendorff, V., Guasch, G., Isnardon, D., Galindo, R., Birn- Khokha, M.K. (2012). Exon capture and bulk segregant anal- baum, D., and Pe´busque, M.J. (1999). Characterization of ysis: rapid discovery of causative mutations using high- FIM-FGFR1, the fusion product of the myeloproliferative dis- throughput sequencing. BMC Genomics 13, 649. order-associated t(8;13) translocation. J. Biol. Chem. 274, 20. del Viso, F., and Khokha, M. (2012). Generating diploid em- 26922–26930. bryos from Xenopus tropicalis. Methods Mol. Biol. 917,33–41. 36. Bekheirnia, M.R., Bekheirnia, N., Bainbridge, M.N., Gu, S., Co- 21. J. Faber and P.D. Nieuwkoop, eds. (1994). Normal Table of ban Akdemir, Z.H., Gambin, T., Janzen, N.K., Jhangiani, S.N., Xenopus laevis (Daudin) (Garland Science), p. 418. Muzny, D.M., Michael, M., et al. (2017). Whole-exome 22. Khokha, M.K., Chung, C., Bustamante, E.L., Gaw, L.W., Trott, sequencing in the molecular diagnosis of individuals with K.A., Yeh, J., Lim, N., Lin, J.C., Taverner, N., Amaya, E., et al. congenital anomalies of the kidney and urinary tract and (2002). Techniques and probes for the study of Xenopus tropi- identification of a new causative gene. Genet. Med. 19, 412– calis development. Dev. Dyn. 225, 499–510. 420. 23. Miller, R.K., Canny, S.G., Jang, C.W., Cho, K., Ji, H., Wagner, 37. Estruch, S.B., Graham, S.A., Quevedo, M., Vino, A., Dekkers, D.S., Jones, E.A., Habas, R., and McCrea, P.D. (2011). Proneph- D.H.W., Deriziotis, P., Sollis, E., Demmers, J., Poot, R.A., and ric tubulogenesis requires Daam1-mediated planar cell polar- Fisher, S.E. (2018). Proteomic analysis of FOXP proteins re- ity signaling. J. Am. Soc. Nephrol. 22, 1654–1664. veals interactions between cortical transcription factors asso- 24. Raciti, D., Reggiani, L., Geffers, L., Jiang, Q., Bacchion, F., Sub- ciated with neurodevelopmental disorders. Hum. Mol. Genet. rizi, A.E., Clements, D., Tindal, C., Davidson, D.R., Kaissling, 27, 1212–1227. B., and Bra¨ndli, A.W. (2008). Organization of the pronephric 38. Giurgiu, M., Reinhard, J., Brauner, B., Dunger-Kaltenbach, I., kidney revealed by large-scale gene expression mapping. Fobo, G., Frishman, G., Montrone, C., and Ruepp, A. (2019). Genome Biol. 9, R84. CORUM: the comprehensive resource of mammalian protein 25. Boualia, S.K., Gaitan, Y., Murawski, I., Nadon, R., Gupta, I.R., complexes-2019. Nucleic Acids Res. 47 (D1), D559–D563. and Bouchard, M. (2011). Vesicoureteral reflux and other uri- 39. Joshi, P., Greco, T.M., Guise, A.J., Luo, Y., Yu, F., Nesvizhskii, nary tract malformations in mice compound heterozygous for A.I., and Cristea, I.M. (2013). The functional interactome Pax2 and Emx2. PLoS ONE 6, e21529. landscape of the human histone deacetylase family. Mol. 26. Narlis, M., Grote, D., Gaitan, Y., Boualia, S.K., and Bouchard, Syst. Biol. 9, 672. M. (2007). Pax2 and pax8 regulate branching morphogenesis 40. Yang, F., Huang, X., Zang, R., Chen, J., Fidalgo, M., Sanchez- and nephron differentiation in the developing kidney. J. Am. Priego, C., Yang, J., Caichen, A., Ma, F., Macfarlan, T., et al. Soc. Nephrol. 18, 1121–1129. (2020). DUX-miR-344-ZMYM2-Mediated Activation of 27. Coyaud, E., Mis, M., Laurent, E.M., Dunham, W.H., Couzens, MERVL LTRs Induces a Totipotent 2C-like State. Cell Stem A.L., Robitaille, M., Gingras, A.C., Angers, S., and Raught, B. Cell 26, 234–250.e7. (2015). BioID-based Identification of Skp Cullin F-box (SCF) 41. Guzzo, C.M., Ringel, A., Cox, E., Uzoma, I., Zhu, H., Black- b-TrCP1/2 E3 Ligase Substrates. Mol. Cell. Proteomics 14, shaw, S., Wolberger, C., and Matunis, M.J. (2014). Character- 1781–1795. ization of the SUMO-binding activity of the myeloprolifera- 28. Kessner, D., Chambers, M., Burke, R., Agus, D., and Mallick, P. tive and mental retardation (MYM)-type zinc fingers in (2008). ProteoWizard: open source software for rapid prote- ZNF261 and ZNF198. PLoS ONE 9, e105271. omics tools development. Bioinformatics 24, 2534–2536. 42. Aguilar-Martinez, E., Chen, X., Webber, A., Mould, A.P., Sei- 29. Craig, R., and Beavis, R.C. (2004). TANDEM: matching proteins fert, A., Hay, R.T., and Sharrocks, A.D. (2015). Screen for with tandem mass spectra. Bioinformatics 20, 1466–1467. multi-SUMO-binding proteins reveals a multi-SIM-binding 30. Eng, J.K., Jahan, T.A., and Hoopmann, M.R. (2013). Comet: an mechanism for recruitment of the transcriptional regulator open-source MS/MS sequence database search tool. Prote- ZMYM2 to chromatin. Proc. Natl. Acad. Sci. USA 112, omics 13, 22–24. E4854–E4863. 31. Teo, G., Liu, G., Zhang, J., Nesvizhskii, A.I., Gingras, A.C., and 43. Wang, J., Liu, M., Zhao, L., Li, Y., Zhang, M., Jin, Y., Xiong, Q., Choi, H. (2014). SAINTexpress: improvements and additional Liu, X., Zhang, L., Jiang, H., et al. (2019). Disabling of nephro- features in Significance Analysis of INTeractome software. genesis in porcine embryos via CRISPR/Cas9-mediated SIX1 J. Proteomics 100, 37–43. and SIX4 gene targeting. Xenotransplantation 26, e12484.

The American Journal of Human Genetics 107, 727–742, October 1, 2020 741 44. Kobayashi, H., Kawakami, K., Asashima, M., and Nishinaka- renal syndrome by disruption of EYA1-SIX1-DNA complexes. mura, R. (2007). Six1 and Six4 are essential for Gdnf expres- Proc. Natl. Acad. Sci. USA 101, 8090–8095. sion in the metanephric mesenchyme and ureteric bud forma- 47. Still, I.H., and Cowell, J.K. (1998). The t(8;13) atypical myelo- tion, while Six1 deﬁciency alone causes mesonephric-tubule proliferative disorder: further analysis of the ZNF198 gene and defects. Mech. Dev. 124, 290–303. lack of evidence for multiple genes disrupted on chromosome 45. Xu, J., and Xu, P.X. (2015). Eya-six are necessary for survival of 13. Blood 92, 1456–1458. nephrogenic cord progenitors and inducing nephric duct devel- 48. Baumann, H., Kunapuli, P., Tracy, E., and Cowell, J.K. (2003). opment before ureteric bud formation. Dev. Dyn. 244,866–873. The oncogenic fusion protein-tyrosine kinase ZNF198/ﬁbro- 46. Ruf, R.G., Xu, P.X., Silvius, D., Otto, E.A., Beekmann, F., blast growth factor receptor-1 has signaling function compa- Muerb, U.T., Kumar, S., Neuhaus, T.J., Kemper, M.J., Raymond, rable with interleukin-6 cytokine receptors. J. Biol. Chem. R.M., Jr., et al. (2004). SIX1 mutations cause branchio-oto- 278, 16198–16208.

742 The American Journal of Human Genetics 107, 727–742, October 1, 2020